This invention relates to a method for reducing Bowman-Birk protease inhibitor levels in plants using either co-suppression or antisense technology.
Seeds of plants contain storage, or reserve, proteins which are synthesized during the development of the seeds. During germination and early seedling growth, these reserves are hydrolyzed to produce metabolic intermediates for use by the growing seedling. In harvested seeds, storage proteins represent an important nutritional source. The nutritional value of these seeds could be increased by altering the composition of the reserve proteins to decrease the amount of undesirable proteins in the seeds.
One major class of proteins found in the seeds of legumes are protease inhibitors which act as regulators of endogenous proteases and as proteins that protect plants from insect and pathogen attack.
Plant protease inhibitors are generally low molecular weight proteins, are characterized by the ability to combine with particular animal, and occasionally plant proteases, thereby abolishing the activity of these enzymes. Research suggests that active protease inhibitors may be toxic to humans and other animals, adversely affecting the nutritional quality of plant foodstuffs, even though they may be beneficial under other circumstances. Thus, there is a need to reduce the level of protease inhibitors in food.
Protease inhibitors are abundant in the legume family and constitute about 6% of the proteins of soybeans. Their antinutritional nature leads to pancreatic hyperplasia, acinar adenoma, and overall growth reduction when raw soybean meal is fed to monogastric animals, such as chicks, rats and quail.
Soybean (Glycine max) seed proteins are one example of storage proteins that are widely used in human foods such as infant formulas, tofu, soy protein isolates, soy flour, textured soy fibers and soy sauce. Soybean protein products serve as an excellent source of low cost, high quality protein for human needs. Soybeans are also widely used as a component of animal feeds. However, they must be properly processed to remove or deactivate protease inhibitors.
Soybean protease inhibitors are categorized into three classes: Kunitz trypsin inhibitors (KTi), Bowman-Birk inhibitors (BBi), and glycine-rich soybean trypsin inhibitors (GRSTi). The amino acid sequence of these inhibitors consists largely of sulfur-containing (methionine and cysteine) amino acids.
The major and predominantly expressed form of KTi is a 21.5 kDa protein which has inhibitory specificity for trypsin. BBi is a low molecular weight (8000 kDa) protein that inhibits both trypsin and chymotrypsin simultaneously at independent reactive sites. At least ten different isoforms of BBi have been reported. GRSTi are minor inhibitors of trypsin in soybean seed.
Various approaches have been taken to reduce the protease inhibitor content and/or activity of soybeans in order to improve the nutritional value of the grain. These include physical (heat) and chemical treatment of soy products, as well as genetic alteration of soybeans through conventional breeding techniques.
In any heat treatment, care must be taken because, even though heating is required to destroy the trypsin inhibitors, improper heating will decrease the nutritional value of the soybean protein itself. Furthermore, although the protease inhibitor activity is largely inactivated by denaturation through conventionally applied heat treatment of soy flour, 10-15% residual activity usually remains. The unusual structure of the BBi is the most likely reason for this residual activity. BBi is strongly cross-linked by disulfide bonds which gives the individual molecules resistance to heat denaturation. Thus, heat treatment of seed or soy products to reduce inhibitor expression is not completely successful and furthermore, is costly in energy usage. Furthermore, soy protein products often contain between 5% and 20% of the activity found in raw beans (Rackis, J. J.; Gumbmann, M. R., in Antinutrients and natural toxicants in food; Ory, R. L., ED.; Food and Nutrition Press; Westport, Conn., 1981, p 203) and there is a concern that this residual activity, due to non-denatured BBi, may be a significant health issue (Liener, I. E. (1986) J. Nutr. 116:920-923).
Another process used to eliminate protease inhibitors from raw soybeans is the solvent-extraction method. This chemical extraction, while removing the various inhibiting materials also results in considerable loss of the oil in the seed, thus, reducing its food value. At the same time, the solvent poses problems of cleanup and disposal.
Genetic modification of the soybean plant to develop low inhibitor activity varieties has also been proposed. However, this idea suffers from a number of drawbacks. Desirable nutritional value may be lost concomitantly with the reduction of the inhibitors. Cross pollination of the genetic variant with another cultivar could result in reexpression of the protease inhibitor gene. Further, altering expression of one inhibitor may not affect the expression of another. As yet, conventional breeding and tissue culture technology has been unable to produce a soybean plant with low levels of protease inhibitors although a need exists for such plants.
The need to reduce the protease inhibitor activity and the high cost of heat deactivation has led to the search of soybean germplasm accessions to find lines that lack the protease inhibitors. Isolines lacking KTi have been developed (Hymowitz, T. (1986) in Nutritional and Toxicological Significance of Enzyme Inhibitors in Foods, M. Friedman ed. Plenum Press. New York.). However, no nulls for BBi have been found in the 12,690 soybean accessions screened to date (Domagalski, J. M. et al. (1992) Crop Sci. 32:1502-1505). BBi nulls have been found in wild perennial Glycine species, however, these species are not traditionally used in a soybean breeding program due to hybridization problems and early pod abortion (Domagalski, J. M. et al. (1992) Crop Sci. 32:1502-1505).
Another approach taken to reduce the level of protease inhibitors in soybean is to purposely express the Brazil Nut storage protein. This protein is high in sulfur-containing amino acids, thus, its expression should limit the availability of this amino acids inhibiting the expression of the protease inhibitors which are also high in sulfur-containing amino acids (WO 95/27068). This method is thought to work because the pool of sulfur-containing amino acids common to both proteins is limiting during seed development. The expression of the new protein effectively competes for the limiting pool of sulfur-containing amino acids resulting in a reduction of the protease inhibitors. Although effective, there are some inherent limitations. Environmental conditions which favor increased sulfur reduction and synthesis of sulfur containing amino acids would cause inhibitor levels to rise. As growing conditions can not always be predicted, the tight control of inhibitor suppression needed for commercial success can not be guaranteed. Also, soybean breeders are trying to increase methionine and cysteine contents in seeds to improve their nutritional value. By doing so, they will select for lines with increased capacity to synthesize methionine and cysteine. This again, will lead to the breakdown of the suppression of the protease inhibitors. In addition, expression of novel high sulfur containing proteins, at levels required for suppression of the protease inhibitors, could lead to the introduction of a potentially new allergenic proteins in the soybean seeds, requiring special considerations for handling and labeling of protein products.
With the availability of KTi null line, genetic modification of soybean plants to develop lines with reduced levels of BBi is desirable. Not only would this decrease the heat required to deactivate protease inhibitor activity and lower residual activity in protein products on its own, but when bred with existing KTi null lines, one could create lines with very low overall protease activity.
BBi contains independent binding sites for both trypsin and chymotrypsin (Liener, I. E., in R. J. Summerfield and A. H. Bunting (eds), Advances in Legume Science, Royal Bot. Gardens, Kew, England). At least 10 isoforms of BBi have been purified from soybean seeds. However, several of the isoforms are related by proteolytic modification and it has been estimated that there are three subgroups of the inhibitor coded for by three independent genetic loci (Tan-Wilson et al. (1987) J. Agric. Food. Chem. 35:974-981). A search of GenBank for BBi sequences of Soybean (Glycine Max) identifies sequences for the classical BBi identified by Bowman and two isoforms designated C II and D II. These sequences are 73% and 83% identical and the encoded amino acid sequences are 57% and 75% identical to the sequences of the gene and protein of the Bowman isoform.
Thus, there remains a need to provide a novel method of eliminating BBi and its isoforms from soybean seeds using either co-suppression or antisense technology.
This invention concerns a method for reducing the level of a Bowman-Birk protease inhibitor in a plant which comprises
(a) transforming plant cells with a chimeric gene comprising a nucleic acid fragment encoding a Bowman-Birk protease inhibitor or a subfragment thereof or complement thereof which is operably linked to a promoter;
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) to obtain seeds; and
(c) selecting from the seeds of step (b) those seeds containing reduced levels of the Bowman-Birk protease inhibitor when compared to plants not containing the chimeric gene.
Also of interest are plants produced by this method which contain reduced levels of a Bowman-Birk protease inhibitor as well as seeds obtained from such plants.
In still another aspect, this invention concerns crossing a plant produced by this method with a plant which is null for a Kunitz trypsin inhibitor and seeds obtained from such a plant.
In still a further aspect, this invention concerns a hybrid plant by crossing a plant made using the instant method with any other parental line wherein the resulting progeny contains in its genome a chimeric gene comprising a nucleic acid fragment encoding a Bowman-Birk protease inhibitor or a subfragment thereof or complement thereof which is operably linked to a promoter.